The present invention relates to a process for the preparation of thermoprecipitating affinity polymers. More particularly, the present invention relates to a process for the preparation of polymers useful for the separation of enzymes of protease type exemplified by trypsin. Affinity polymers prepared by the process of the present invention exhibit stronger binding with trypsin which is useful in enhancing the recovery of trypsin from dilute aqueous solutions and from a mixture of trypsin and chymotrypsin or a mixture of trypsin and other enzymes.
Isolation and purification of biologically active macromolecules such as enzymes, from natural sources is a tedious, multi-step process, which results in very low yields and thus higher costs. As a better alternative to conventional processes, researchers have developed affinity separations based techniques for selective and enhanced separations of enzymes. The basic principle used in these techniques is to form a complex between the active site of an enzyme and inhibitor, selective and high separations are possible. Most of the affinity based operations involved polymers to which inhibitors are chemically linked. The complex formed between polymeric inhibitor and the enzyme is subsequently processed to isolate the enzyme.
Various techniques such as affinity chromatography, affinity partitioning, affinity ultrafiltration, immobilized metal affinity chromatography, affinity imprinting and affinity precipitation have been developed so far, Although all these techniques use the same basic principle of forming an enzyme-inhibitor complex, they suffer from one or the other disadvantages as follows.
Affinity chromatography uses a column containing an inhibitor or a dye or an antibody for a given enzyme for its separation from a mixture of enzymes. The solution of enzymes is poured over the affinity column to retain the desired enzyme on column for subsequent isolation. This technique is efficient only for small capacity columns. With the scale up of columns, the problems of sample pre-treatment and plugging of packed column becomes severe. [Y. Li, G. Kunyu, C. Lubai, Z. Hanfa, Z. Yunkui Sepu, 14, 415 (1996), T. Makriyannis, Y. D. Clonis, Biotech. Bioengg. 53, 49 (1997)].
In case of affinity crossflow ultrafiltration, a mixture of enzymes is filtered through a membrane containing affinity group under pressure. This technique is suitable in the cases where the difference between the molecular weights. of the two enzymes is high. Also,with the increase-in the filtration time, denaturation of enzymes as well as clogging of membrane takes place due to the pressure applied. [K. Sigmundsen, H. Filippusson, Polymer Int. 41, 335 (1996); T. B. Choe, P. Masse, A. Verdier, Biotech.Lett., 8, 163 (1986)].
Affinity partitioning of two-phase aqueous systems is widely used technique as compared to the methods mentioned above. In this technique, concentrated aqueous solution of poly (ethylene glycol) (PEG) with or without linking affinity group is mixed with enzyme solution containing moderate to high salt concentration. The two phases are mixed well and allowed to separate. The desired enzyme gets predominantly partitioned in one phase, which subsequently can be isolated. Disadvantages of this technique are non-specific extraction of other proteinaceous molecules along with desired enzyme and also poor interactions between enzyme and affinity group due to high ionic strength. [G. Takerkart, E. Segard, M. Monsigny, FEBS Lett., 42, 218 (1972), B. A. Andrews, D. M. Head, P. Dunthorne, J. A. Asenjo, Biotech. Tech., 4, 49 (990)].
Immobilised metal affinity chromatography is a technique in which the columns of polymeric support containing chelated metal ions are used. These metal ions form coordination complex with histidine, tyrosine, cysteine, etc. present on the surface of the enzyme. Although this technique has advantages like high column capacity, ease in enzyme elution, etc. it is not very selective. [Ehteshami, J. Porath, R. Guzman, G. Ehteshami, J. Mol. Recognit. 9, 733 (1996); A. L. Blomkalns, M. R. Gomez, Prep. Biochem. Biotechnol. 27, 219 (1997)].
Molecular imprinting of matrices containing metal, chelates is a recently developed technique, which increases the selectivity [F. H. Arnold, P. Dahl, D. Shnek, S. Plunkett, U.S. Pat. No. 5,310,648 (1994)]. In this technique complex of monomer containing chelated metal ion and enzyme is polymerised with crosslinker in order to imprint the polymer with enzyme. Although this technique exhibits a substantial selectivity, it is not as selective as that of biological antibodies or active site inhibitors of enzymes.
Compared to the techniques described above, affinity precipitation is an attractive technique from the point of view of application. [C. Senstad, B. Mattiasson, Biotech.Bioengg., 33, 216 (1989); M. Schneider, C. Guillot, B. Lamy, Ann. N.Y. Acad Sci. 369, 257 (1981); B. Mattiasson, R. Kaul, xe2x80x9cAffinity precipitationxe2x80x9d, in Molecular interactions in bioseparations, T. T. Ngo ed., Plenum Press, New xe2x80x9cYork, p 469-477 (1993), J. P. Chen, J. Ferment and Bioengg., 70, 119 (1990); I. Y. Galaev, B. Mattiasson, Biotech. Bioeng. 41, 1101 (1993); M. Pecs, M. Eggert, K. Schnegerl, New Polymeric Mater. 4, 19 (1993)]. It involves formation of complex between an enzyme and a stimuli sensitive polymeric inhibitor. This complex is precipitated by pH or temperature stimulus and isolated. It is then dissociated, polymer separated by pH or temperature stimulus and the enzyme isolated. Thus, the recovery of the enzyme by this technique is much simpler and the scale up. of the process is also easy. Hitherto, affinity precipitation suffers from restrictions on the accessibility of the enzyme towards the polymer bound inhibitor.
The strength of the complex formed between inhibitor and the enzyme decreases 20 to 300 fold when it is bound to the polymer. [K. B. Male, J. H. T. Luong, A. L. Nbuyen, Enzy. Microb. Tech., 9, 374 (1987); Yu, I. Galled, B. Matisson, Biotech. Bioengg. 41, 1101 (1993); J. H. T. Loung, K. B. Male, A. L. Nguyen, Biotech Bioengg., 31, 439 (1988); M. Pecs, M. Eggert, K. Schuegerl, J. Biotech., 21, 137 (1991)]. This weakening of the complex is attributed mainly to the restrictions on the free access of enzyme to the polymer bound inhibitor. The strength of the complex is expressed in terms of inhibition constant (Ki). The lower the value of Kj the higher is the inhibition and stronger is the complex formed. Higher Ki values of polymeric inhibitors result in poor recovery of enzymes. Also, increased concentration of inhibitors on the high molecular weight polymers results in high Kj values.
Introduction of spacers between the polymer backbone and the inhibitor is a well-known methodology used in affinity chromatography to enhance the interaction between inhibitor and the enzyme. But in affinity precipitation, the use of spacer containing polymers has not been reported so far, because the complex formation between polymer bound inhibitor and the enzyme takes place in homogeneous solution and it has been suggested that in homogeneous solutions spacers are not required.
It is therefore an object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers comprising spacers between the polymer backbone and the inhibitor, useful in enhanced recovery process of trypsin by affinity precipitation.
It is another object of the invention to provide a process for the preparation of thermoprecipitating affinity polymers that exhibit enhanced interactions with the enzymes and thereby give high recovery for the desired enzymes.
Accordingly the present invention provides a process for the preparation of thermoprecipitating affinity polymers useful in the enhanced recovery of enzymes which comprises polymerising a monomer comprising a spacer and a co-monomer with a polymerisation initiator and a polymerisation accelerator at ambient temperature and pressure for a period ranging between 2 to 24 hours to obtain a polymer, linking an inhibitor to pendant carboxyl groups of the spacers in the polymer to obtain an affinity polymer by any conventional method.
The spacer monomer may be selected from compounds of the formula CH2xe2x95x90CRxe2x80x94COxe2x80x94NHxe2x80x94(CH2)nxe2x80x94COOH, wherein R is hydrogen or methyl group and n is an integer between 1 to 10.
The comonomer may be N-isopropyl acrylamide, N-butyl acrylamide, N-isopropyl methacrylamide or N-vinyl caprolactam.
The molar ratio of spacer monomer to co-monomer may be from 1:10 to 1:1.
The polymerisation initiator may be compounds such as ammonium persulfate or potassium persulfate.
The polymerisation initiator may be 10% to 20% based on the weight of the monomers.
The polymerisation accelerator may be selected from compounds such as N,N,Nxe2x80x2,Nxe2x80x3tetramethyl ethylene diamine, sodium meta bisulfate or potassium meta bisulfate.
The polymerisation accelerator is 1% to 5% based on the weight of the monomers.
The inhibitor may be meta amino benzamidine, para amino benzamidine or their hydrochlorides.
The molar ratio of inhibitor to carboxyl groups is from 1:1 to 10:1.
The condensing reagent used for linking the inhibitor to the pendant carboxyl groups of the polymer is, for example, 1-cyclohexyl 3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulphate (CMC) or 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC).
The molar ratio of the condensing agent to carboxyl groups may be from 1:1 to 100:1.
The thermoprecipitating affinity polymers comprising spacers is typically prepared under mild conditions by dissolving the spacer monomer, co-monomer and polymerisation initiator in water and purging the solution with nitrogen for 10 to 20 minutes. The polymerisation accelerator is added and the solution is kept at 37xc2x0 C. for 24 hours for polymerisation. After polymerisation, temperature of the solution is raised above lower critical solution temperature (LCST) of the polymer and precipitated polymer is isolated.
In another feature of the inventions the polymer is dissolved in water at 10xc2x0 C. One to ten fold molar excess of inhibitor and condensing reagent over the carboxyl groups in the polymer is added to this solution. The solution is stirred for 1 to 12 hours at 10xc2x0 C. Inhibitor-linked polymer, i.e. affinity polymer is then precipitated by raising the temperature above its LCST (37 to 65xc2x0 C.) and precipitated affinity polymer is isolated.
The affinity polymers synthesised by the process of the present invention are used in trypsin recovery. A solution of the affinity polymer is mixed with a solution of trypsin and chymotrypsin and allowed to stand at 4 to 25xc2x0 C. for 15 minutes to 1 hour. The temperature of the solution is then raised above the LCST of the affinity polymer (37 to 65xc2x0 C.) to precipitate the polymer-trypsin complex. This complex is separated by centrifugation and the polymer-trypsin complex is dissociated by dissolving it in an acidic buffer. The temperature of the solution is then raised above LCST of the affinity polymer. The polymer is separated by centrifugation and the clear filtrate exhibiting trypsin activity is isolated.
Although the present invention describes a process for the preparation of thermoprecipitating affinity polymers useful in the enhanced -recovery of trypsin from a mixture of trypsin and chymotrypsin, the scope of the present invention is not and should not be construed to limit to only such affinity polymers for the separation of trypsin. It may extend to such combinations of polymer: bound inhibitors and their respective enzymes.
The ranges and limitations provided in the present specification, examples and claims are those believed to particularly point out and distinctly cover the present invention. However, other ranges and limitations which perform substantially the same function in the same or substantially the same manner to obtain the same or substantially the same results are intended to be within the scope of the instant invention.